Bottom Line:
We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres.The stage-dependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions.Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.

Affiliation: Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.

ABSTRACTWe have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stage-dependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.

Figure 7: Analysis of the distribution of distances between telomeres. The Euclidean straight-line distances between all the pairs of telomeres were determined for each nucleus and normalized to the maximum distance, generating a distance distribution curve that can be directly compared for nuclei that differ in volume and telomere signal count (see Materials and Methods). The relative frequencies of these distances were binned by increments of 10% of the maximum (see Materials and Methods). These binned normalized pairwise distance values were averaged to yield a single curve for each stage (stage is indicated in the legend at top, n, number of nuclei averaged). The “Random” curve was made from modeled nuclei as described in Materials and Methods, and is plotted for comparison in both graphs (thick line). (A) The distribution of distances for nuclei before and during the bouquet stage. (B) The distribution of distances during and after the bouquet stage.

Mentions:
Mathematical analyses, based on the spatial X, Y, and Z coordinates of each telomere, were used to objectively quantify the changes in telomere positions that were revealed by inspection of the images and models (Figs. 3–6). The analyses of overall telomere distributions are shown in Figs. 7 and 8 and summarized in Table I. We first analyzed the distribution of distances between the telomeres relative to the diameter of the nucleus. The Euclidean distances between all possible pairs of points in a given volume has a characteristic distribution (27). For comparison with the telomere data, the distribution of distances that characterizes the available space in nuclei with large internal nucleoli were determined as follows. We used a Monte Carlo simulation to generate 5,000 randomly placed points within individual nuclei, but outside the nucleolus (see Materials and Methods). The distribution of pairwise distances was determined for the randomly placed points in order to derive a “random” curve that took into account the presence of the large nucleolus. This allowed us to compare the distribution of telomeres to the distribution of available space as shown in Fig. 7. The distributions of pairwise distances for telomeres in nuclei preceding the bouquet stage are similar to each other, and when compared to the random curve, show a tendency to have greater than expected numbers of large distances (Fig. 7 A). In striking contrast, the clustering of telomeres at the bouquet stage is clearly evident in the dramatic shift towards smaller values in the pairwise distances curve. The dispersal of telomeres after the bouquet stage is revealed in Fig. 7 (B) by the shift in the pairwise distances curve back towards the larger pairwise distance categories. The proximity of homologous telomeres in paired chromosomes (as seen for example in Fig. 2 C), is reflected in the greater than expected frequency of short distances at the left-most region of the curve (compare the values at different stages for the smallest pairwise distance category, 10%). These data indicate that the telomere cluster is not formed by a gradual coming together of telomeres during premeiotic interphase and leptotene, but rather by a relatively abrupt mechanism.

Figure 7: Analysis of the distribution of distances between telomeres. The Euclidean straight-line distances between all the pairs of telomeres were determined for each nucleus and normalized to the maximum distance, generating a distance distribution curve that can be directly compared for nuclei that differ in volume and telomere signal count (see Materials and Methods). The relative frequencies of these distances were binned by increments of 10% of the maximum (see Materials and Methods). These binned normalized pairwise distance values were averaged to yield a single curve for each stage (stage is indicated in the legend at top, n, number of nuclei averaged). The “Random” curve was made from modeled nuclei as described in Materials and Methods, and is plotted for comparison in both graphs (thick line). (A) The distribution of distances for nuclei before and during the bouquet stage. (B) The distribution of distances during and after the bouquet stage.

Mentions:
Mathematical analyses, based on the spatial X, Y, and Z coordinates of each telomere, were used to objectively quantify the changes in telomere positions that were revealed by inspection of the images and models (Figs. 3–6). The analyses of overall telomere distributions are shown in Figs. 7 and 8 and summarized in Table I. We first analyzed the distribution of distances between the telomeres relative to the diameter of the nucleus. The Euclidean distances between all possible pairs of points in a given volume has a characteristic distribution (27). For comparison with the telomere data, the distribution of distances that characterizes the available space in nuclei with large internal nucleoli were determined as follows. We used a Monte Carlo simulation to generate 5,000 randomly placed points within individual nuclei, but outside the nucleolus (see Materials and Methods). The distribution of pairwise distances was determined for the randomly placed points in order to derive a “random” curve that took into account the presence of the large nucleolus. This allowed us to compare the distribution of telomeres to the distribution of available space as shown in Fig. 7. The distributions of pairwise distances for telomeres in nuclei preceding the bouquet stage are similar to each other, and when compared to the random curve, show a tendency to have greater than expected numbers of large distances (Fig. 7 A). In striking contrast, the clustering of telomeres at the bouquet stage is clearly evident in the dramatic shift towards smaller values in the pairwise distances curve. The dispersal of telomeres after the bouquet stage is revealed in Fig. 7 (B) by the shift in the pairwise distances curve back towards the larger pairwise distance categories. The proximity of homologous telomeres in paired chromosomes (as seen for example in Fig. 2 C), is reflected in the greater than expected frequency of short distances at the left-most region of the curve (compare the values at different stages for the smallest pairwise distance category, 10%). These data indicate that the telomere cluster is not formed by a gradual coming together of telomeres during premeiotic interphase and leptotene, but rather by a relatively abrupt mechanism.

Bottom Line:
We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres.The stage-dependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions.Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.

Affiliation:
Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.

ABSTRACTWe have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stage-dependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.